ABSTRACT Type 1 diabetes (T1D) is an autoimmune disease caused by lymphocytes attacking self- antigens of islets. At present, there is no safe and effective therapy for treating T1D in humans. The imbalance of protective regulations and self-reactive immune reactions results in the disease. Both diabetic patients and animals have malfunctioning and/or decreased number of regulatory T cells (Tregs). Diabetes self-antigen specific Tregs can selectively travel to pancreas and inhibit disease with 200-fold higher efficacy than non-antigen specific Tregs. It has been very difficult to identify antigen specific Tregs from diabetic patients or mice, correct their function and grow large number of good Tregs to treat disease. Thus, generating functional antigen-specific Tregs to prevent or reversal T1D is a promising method. We generated antigen specific Tregs, and we propose to test their ability to prevent and reverse diabetes using spontaneous T1D mouse model in this application. Previously we discovered the T1D disease-causing insulin antigen, named insulin B chain register #3 (InsuinB:R3), which is different from the hormone insulin. We also generated an antibody, mAb287, that blocks the InsulinB:R3 and prevents T1D partly. Chimeric Antigen Receptor redirected T cell (CAR- T) technique grants antigen specificity onto T cells, and this technique has been used in treating blood cancer successfully. Thus, we generated mAb287-CAR-CD8 T cells and mAb287-CAR-Tregs. We already reported that the mAb287-CAR-CD8 T cells recognize the T1D-causing InsulinB:R3, and selectively travel to pancreas. Tregs specific for one antigen can suppress immune responses induced by multiple antigens which is usually the case in the development of T1D. Thus, we hypothesize that mAb287-CAR-Tregs is a safe immune therapy to prevent and reverse T1D in mice and will test it with two Aims. 1. To evaluate the protective phenotype of mAb287-CAR-Tregs and their stable survival at pancreas. We will stimulate mAb287-CAR-Tregs with antigens to test their regulatory function. We will label the mAb287-CAR-Tregs with cell-trace-dye to track their location, proliferation and stability in mice. 2. To test the efficacy of mAb287-CAR-Tregs alone or in combination with cell-selected low dose IL-2 treatment to prevent and reverse T1D in mice. We will inject mAb287-CAR-Tregs to pre-diabetes or new-onset diabetic mice, then monitoring the levels of blood glucose. Our goal is to develop a safe and effective antigen specific Treg therapy to treat T1D. Our antigen specific CAR-Treg approach overcomes one barrier of treating diabetes, a lack of antigen specific regulatory T cells. If successful, this therapy is expected to be adopted to human T1D as InsulinB:R3 is an important self-antigen in T1D patients. To facility the translation of our study, we already generated multiple antibodies specific for the human InsulinB:R3 antibodies.